Ceramides and related sphingolipids are key structural components of biological membranes and cell messengers involved in the regulation of cell proliferation, differentiation and numerous differentiated cell functions, senescence and apoptosis. Ceramide is formed by three major pathways: acylation of sphinganine to dihydroceramides followed by desaturation or hydroxylation;acylation of sphingosine;and hydrolysis of more complex sphingolipids, such as sphingomyelin. This grant focuses on the enzymes responsible for acylation of sphingoid bases (termed ceramide synthases) for which we have recently identified three mammalian genes (termed LASS1, 4 and 5 for longevity assurance gene homologs) that each code for (dihydro)ceramide synthases that produces specific molecular subspecies of (dihydro)- ceramide due to a high degree of selectivity for the fatty acyl-CoA co-substrate. In this grant we will further characterize these as well as additional mammalian LASS homologs to determine the roles of each in sphingolipid synthesis. The studies will employ a combination of approaches: use of gene cloning to express, and RNAi to suppress, the genes of interest;biochemical characterization of the ceramide synthase activities;fluorescence microscopy to explore subcellular localization;and liquid chromatography, electrospray ionization tandem mass spectrometry (MS/MS and MS/MS/MS) for in-depth, quantitative analyses of ceramide subspecies, complex sphingolipids and other metabolites of interest (e.g., sphingoid base 1-phosphates, etc.). Mass spectrometric analyses will also be used to quantify the biosynthesis and turnover of specific subspecies using stable isotope labeled backbones. These tools will also be used to explore how ceramide biosynthesis is regulated at the levels of expression of the mRNA for specific LASS family members under conditions where sphingolipid metabolism has been perturbed genetically and (or) by addition of exogenous sphingolipids and analogs, which include the drugs safingol and FTY720. These studies will provide fundamental information about sphingolipid metabolism, and the selected experimental systems will provide insight into the roles of ceramide synthases in diseases such as cancer where there are known abnormalities in LASS1 expression as well as an involvement of ceramide synthase(s) in the action of sphingolipid-based therapeutics.